The objective of this research is to elucidate the control mechanisms involved in the regulation of de novo pyrimidine biosynthesis in mammalian cells. The first three enzymes of the pathway; carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase are associated with a single polypeptide chain. This large multifunctional protein, complex A, also exhibits allosteric transitions. The protein isolated from a mutant SV40 transformed hamster cell line, will be cleaved by controlled proteolysis to determine the number, function and arrangement of autonomously folded structural domains. These studies will employ chemical modification, peptide mapping, end group analysis and crosslinking experiments. The structure and interconversion of the oligomeric forms of the complex will be studied by gel filtration and ultracentrigation. Studied will focus on the unique functional properties such as channeling, reduction in transient time, coordinate effects and catalytic facilitation and the interrelationship of these properties to the allosteric control mechanisms. The kinetics of the system will be simulated by a computer model, to assess the extent to which compartmentation of intermediates can account for the functional properties of the complex. Using a combination of techniques, enzymatic assays, immunoprecipitation, gel electrophoresis and fluorecent microscopy, the activity, oligomeric form, intracellular concentration and location and the turnover of the molecule will be studied in cultured cells.